The ubiquitin system is the linchpin in maintenance of cellular fitness. While many studies have focused on ubiquitylation pathways, comparatively little is known about deubiquitination proteins (DUBs). DUBs are a large group of proteases that regulate ubiquitin-dependent regulatory pathways by cleaving ubiquitin-protein bonds. DUBs can also cleave C-terminally modified ubiquitin. DUBs are also commonly referred to as deubiquitinating proteases, deubiquitylating proteases, deubiquitylating proteinases, deubiquitinating proteinases, deubiquitinating peptidases, deubiquitinating isopeptidases, deubiquitylating isozpeptidases, deubiquitinases, deubiquitylases, ubiquitin proteases, ubiquitin hydrolyases, ubiquitin isopeptidases, or DUBs. The human genome encodes in five gene families nearly 100 DUBs with specificity for ubiquitin. Importantly, DUBs may act as negative and positive regulators of the ubiquitin system. In addition to ubiquitin recycling, they are involved in processing of ubiquitin precursors, in proofreading of protein ubiquitination, and in disassembly of inhibitory ubiquitin chains. The term DUBs also commonly refers to proteases that act on ubiquitin-like proteins such as SUMO, NEDD and ISG15. Such DUBs are also known as deSUMOylases, deNEDDylases and deISGylating.
DUBs play several roles in the ubiquitin pathway. First, DUBs carry out activation of ubiquitin and ubiquitin-like proproteins. Second, DUBs recycle ubiquitin and ubiquitin-like proteins that may have been accidentally trapped by the reaction of small cellular nucleophiles with the thiol ester intermediates involved in the ubiquitination of proteins. Third, DUBs reverse the ubiquitination or ubiquitin-like modification of target proteins. Fourth, DUBs are also responsible for the regeneration of monoubiquitin from unanchored polyubiquitin, i.e., free polyubiquitin that is synthesized de novo by the conjugating cellular machinery or that has been released from target proteins by other DUBs. Finally, the deubiquitinating enzymes UCH-L3 and YUH1 are able to hydrolyse mutant ubiquitin UBB+1 despite the fact that the glycine at position 76 is mutated.
One of the main classes of DUBs is cysteine protease DUBs, examples of which include members of the ubiquitin-specific processing protease (USP/UBP) superfamily, and members of the ubiquitin C-terminal hydrolyase (UCH) superfamily. In humans, these proteases are involved in processes including apoptosis, autophagy, cell cycle, DNA repair, chromosome remodeling, transcription, endocytosis, MHC class II immune responses, cytokine responses, oxidative stress response, angiogenesis, metastasis, prohormone processing, and extracellular matrix remodeling important to bone development. Because the ubiquitin pathways are involved in many important physiological processes, the DUBs are potential targets for the treatment of many diseases, including cancer, inflammation, neurodegeneration, and infection.
Cysteine proteases are potential targets for the treatment of many diseases, including inflammation, spinal cord injury, neurodegeneration, autoimmune diseases, infection, and cancer. A general strategy for the design of cysteine protease inhibitors consists of identification of a “warhead” functionality that reacts with the catalytic cysteine, and recognition elements that target specific inhibitors. Most “warheads” are very reactive functionalities, such as Michael acceptors, epoxides and haloketones, that often react nonspecifically with other proteins. There exists a need for new warheads with lower intrinsic activity and the ability to temporarily modify their targets.
Currently-available cell permeable small molecule inhibitors of DUBs, such as G5 and NSC632839, are reactive compounds that irreversibly modify other proteins in addition to DUBs. Many known DUB inhibitors have two reactive sites that will non-specifically cross-link proteins, causing an accumulation of both high molecular weight ubiquitin species and protein aggregates in in vitro assays. Thus, there exists a need for inhibitors of DUBs or cysteine proteases with reduced intrinsic reactivity.